Microscopy-based assays of cell function provide important insights into human immunology and disease that are not attainable through other forms of analysis. Importantly, contemporary microscopy platforms are well suited for providing detailed spatial and functional information from small numbers of cells, a strategic property when the cells of interest are of limited frequency or availability. Unfortunately, loss of cells i a sample to dead volume spaces during sample preparation and handling, particularly when engineered surfaces are used to test cell function, severely limits the realization of this potentil. To address this low efficiency of cell analysis, we seek to develop two complementary microscopy slide chambers that allow the use of complex model systems for cell stimulation while increasing the efficiency of cell loading. The first system incorporates a modified well design that focuses cell seeding onto a small substrate area on which cell imaging and analysis takes place. The second system uses magnetic capture to localize cell seeding onto a similarly specified area, and is well suited for systems in which positive selection is used to prepare cells The goal of this project is to transition from the flexible but low-throughput methods used to fabricate the initial prototypes to higher-throughput systems. Specifically, we will transition fro elastomer-based soft lithography to injection molding of plastics commonly used in conventional labware. As a specific application of these chambers, we will examine the ability of regulatory T cells to respond to the microscale organization of TCR/CD28/LFA-1 stimulation. Successful completion of the proposed project will provide a new assay platform that is well suited for cellular and molecular studies of immune function. Strategic deployment of these systems throughout the research community will greatly accelerate the translation of basic research into clearer understanding of immune-based diseases.